The broad objective of this project is to elucidate the role of cytotoxic effector mechanisms in the evolution of alloimmune responses. Proposed studies will focus on the role of the granule thiol protease, dipeptidyl peptidase I (DPPI), in both the generation of cytotoxic T lymphocyte (CTL) effector function and in the regulation of acute and chronic T cell responses. Prior studies supported by this project were the first to recognize that DPPI is expressed at high levels in the specialized effector granules of CTL, natural killer cells, mast cells and neutrophils where this enzyme is responsible for post-translational processing and activation of the granule serine proteases expressed by these immune effector cells. Inhibition of DPPI function by selective protease inhibitors or deletion of DPPI expression by targeted mutations in the DPPI gene abolishes processing and activation of Granzyme A and Granzyme B and prevents induction of target cell apoptosis by perforin/granzyme dependent mechanisms. Preliminary studies indicate that DPPI deficient and perforin deficient CTL exhibit similar defects in induction of target cell apoptosis, but DPPI and perforin deficiency have distinctly different effects on CD8+ T cell proliferation and expansion during immune responses. DPPI deficiency and/or inhibition but not perforin deficiency is associated with reduced initial CD8+ T proliferative responses to sub-maximal stimuli. In addition, dramatically increased expansion of Tc1 cytokine producing CD8+ T cells is observed after transfer of perforin deficient CD8+ T cells but not after transfer of DPPI deficient CD8+ T cells to MHC Class I disparate hosts. Proposed future studies will examine the mechanisms underlying these differences in effects of DPPI versus perforin deficiency on T cell function. We also will address the hypothesis that DPPI deficiency likely has different effects than perforin deficiency on the evolution of graft versus host disease (GVHD). These latter studies will examine the role of perforin/granzyme cytotoxic effector mechanisms in mediating liver and intestinal GVHD and should provide new insight into the role that such effector mechanisms play in the complications of bone marrow and organ transplantation.